OC.13.3: Metformin Inhibits Proliferation, Enhances Apoptosis and Down-Regulates Epithelial to Mesenchymal Transition (EMT) in Human Cholangiocarcinoma (CCA): A Study on Human Primary Cell Cultures

CCA is a very aggressive cancer with marked resistance to chemotherapeutics. We havepreviously demonstrated that CCA is enriched of cancer stem cells expressing EMT traits, thesefeatures being associated with aggressiveness and drug resistance. We have recentlyestablished primary cell cultures from human intrahepatic CCA subtypes (i.e., mucin andmixed). Treatment with the anti-diabetic drug metformin has been recently associated withreduced cancer incidence. Furthermore, in immortalized cancer cell lines, metformin showedinhibitory effects on EMT by up-regulating Foxo3a signaling in an Akt-dependent manner. Weaimed to evaluate the effects of metformin on proliferation, apoptosis, cell migration and theexpression of EMT traits in primary cultures of CCA subtypes.Primary CCA cell cultures were treated with increasing metformin concentrations (from 5 to1000 µM, for 1-4 days). Then we evaluated: (i) proliferation by MTS assay; (ii) apoptosis byflow cytometry analysis of Annexin VFITC/Propidium Iodide (PI); and (iii) cell migration bywound-healing assay. The expression of Vimentin, E-Cadherin, SNAIL1, SNAIL2, TWIST1,Cytokeratin19(CK19), FOXO3a and AMPK genes were analyzed by RT-qPCR, whereas ck19,Vimentin, E-Cadherin and Foxo3a were analyzed by Immunofluorescence Assay.Metformin inhibited cell proliferation (MTS assay, population doubling and population doublingtime) and induced apoptosis in primary cultures of mucin- and mixed-CCA; the effects weredose- and time-dependent (p<0.05 vs. controls). The migration of primary human CCA cells,from both mucin and mixed CCA subtypes, was also significantly reduced by treatment withmetformin at different concentrations, from 5 to 1000 µM. The effects of metformin wereassociated with enhanced gene expression of the epithelial marker E-Cadherin and decreasedexpression of Vimentin and EMT specific genes, SNAIL1, SNAIL2 and TWIST1. Metformin alsoincreased the AMPK and Foxo3a mRNA levels. FOXO3a gene expression was negativelycorrelated with the expression of SNAIL1 and Vimentin genes. Metformin exerted similareffects in mucin- and mixed-CCA human primary cell cultures.In conclusion, we demonstrated that metformin inhibits cell proliferation, enhances apoptosisand impairs the expression of EMT traits by upregulating Foxo3a gene in primary cultures ofhuman CCA. Therefore, metformin could play anticancer effects against human CCAs withrelevant therapeutic implications.